Our aim is to develop a DNA sequencing device based upon the blockade of ionic current in the transmembrane protein pore a-hemolysin (aHL). Compared to other proposed nanopore-based approaches, the protein pore current blockade (PPCB) method is arguably the simplest, both conceptually and technologically, but recently has been passed over in favor of more complex methods. Recent electronic readout and bilayer lipid membrane advances by the proposers have greatly alleviated prior signal-to-noise ratio and robustness issues. New experimental data and an accurate Stochastic Model for DNA Motion (SMDM) within a-HL now indicate threshold feasibility for sequencing by the PPCB method. Inspection of calculated SMDM responses to known input DNA sequences shows that the principal issue for sequencing via the PPCB method is random variance in the order the bases pass through the pore, leading to three quantifiable sources of error. This program will make specific structural and measurement parameter modifications to the present apparatus to reduce each type of error and to produce a minimal overall sequencing error. The final apparatus will be evaluated by sequencing up to 30 Mbases of natural DNA. PUBLIC HEALTH RELEVANCE: This project offers a path to low cost DNA sequencing. Determining the DNA sequence is useful in basic research studying fundamental biological processes, as well as in applied fields such as diagnostic or forensic research. The advent of DNA sequencing has significantly accelerated biological research and discovery, but current methods are complicated and expensive, thus limiting their applicability. Simple, inexpensive DNA sequencing offers the capability to apply the benefits of the process to everyday medical and forensic applications.